Proteins expressed by mycobacterium tuberculosis and not by BCG and their use as diagnostic reagents and vaccines

ABSTRACT

The present invention is directed to reagents useful for generating immune responses to  Mycobacterium tuberculosis  and for diagnosing infection and disease in a subject that has been exposed to  M. tuberculosis.

This application is a Divisional of U.S. patent application Ser. No. 14/201,308 filed Mar. 7, 2014, now U.S. Pat. No. 9,238,066, which is a divisional of, and claims priority to, U.S. application Ser. No. 13/893,659, filed May 14, 2013, now U.S. Pat. No. 8,974,800, which is a divisional of, and claims priority to, U.S. application Ser. No. 13/198,108, filed Aug. 4, 2011, now U.S. Pat. No. 8,992,942, which is a continuation of, and claims priority to, U.S. application Ser. No. 12/503,717, filed Jul. 15, 2009 and now U.S. Pat. No. 8,021,832, which is a continuation of, and claims priority to, U.S. application Ser. No. 11/677,502, filed Feb. 21, 2007, now U.S. Pat. No. 7,579,141, which is a divisional of, and claims priority to, U.S. application Ser. No. 10/009,383, filed Mar. 4, 2002 and now U.S. Pat. No. 7,932,373, which claims priority to International Patent Application No. PCT/US00/12257, filed May 4, 2000, which claims priority to U.S. Provisional Application Ser. No. 60/132,505, filed May 4, 1999, the disclosures of each of which are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Tuberculosis infection continues to be a world-wide health problem. This situation has recently been greatly exacerbated by the emergence of multi-drug resistant strains of M. tuberculosis and the international AIDS epidemic. It has thus become increasingly important that effective vaccines against and reliable diagnostic reagents for M. tuberculosis be produced.

The disclosure of U.S. Pat. No. 6,087,163 is incorporated herein by reference in its entirety.

SUMMARY OF THE INVENTION

The invention is based on the inventor's discovery that a polypeptide encoded by an open reading frame (ORF) in the genome of M. tuberculosis that is absent from the genome of the Bacille Calmette Guerin (BCG) strain of M. bovis elicited a delayed-type hypersensitivity response in animals infected with M. tuberculosis but not in animals sensitized with BCG. Thus proteins encoded by ORFs present in the genome of M. tuberculosis but absent from the genome of BCG represent reagents that are useful in discriminating between M. tuberculosis and BCG and, in particular, for diagnostic methods (e. g., skin tests and in vitro assays for M. tuberculosis-specific antibodies and lymphocyte responsiveness) which discriminate between exposure of a subject to M. tuberculosis and vaccination with BCG. The invention features these polypeptides, functional segments thereof, DNA molecules encoding either the polypeptides or the functional segments, vectors containing the DNA molecules, cells transformed by the vectors, compositions containing one or more of any of the above polypeptides, functional segments, or DNA molecules, and a variety of diagnostic, therapeutic, and prophylactic (vaccine) methodologies utilizing the foregoing.

Specifically, the invention features an isolated DNA molecule containing a DNA sequence encoding a polypeptide with a first amino acid sequence that can be the amino acid sequence of the polypeptide MTBN1, MTBN2, MTBN3, MTBN4, MTBN5, MTBN6, MTBN7 or MTBN8, as depicted in FIGS. 1A and 1B, or a second amino acid sequence identical to the first amino acid sequence with conservative substitutions; the polypeptide has Mycobacterium tuberculosis specific antigenic and immunogenic properties. Also included in the invention is an isolated portion of the above DNA molecule. The portion of the DNA molecule encodes a segment of the polypeptide shorter than the full-length polypeptide, and the segment has Mycobacterium tuberculosis specific antigenic and immunogenic properties. Other embodiments of the invention are vectors containing the above DNA molecules and transcriptional and translational regulatory sequences operationally linked to the DNA sequence; the regulatory sequences allow for expression of the polypeptide or functional segment encoded by the DNA sequence in a cell. The invention encompasses cells (e.g. eukaryotic and prokaryotic cells) transformed with the above vectors.

The invention encompasses compositions containing any of the above vectors and a pharmaceutically acceptable diluent or filler. Other compositions (to be used, for example, as DNA vaccines) can contain at least two (e. g., three, four, five, six, seven, eight, nine, ten, twelve, fifteen, or twenty) DNA sequences, each encoding a polypeptide of the Mycobacterium tuberculosis complex or a functional segment thereof, with the DNA sequences being operationally linked to transcriptional and translational regulatory sequences which allow for expression of each of the polypeptides in a cell of a vertebrate. In such compositions, at least one (e. g., two, three, four, five, six, seven, or eight) of the DNA sequences is one of the above DNA molecules of the invention. The encoded polypeptides will preferably be those not encoded by the genome of cells of the BCG strain of M. bovis.

The invention also features an isolated polypeptide with a first amino acid sequence that can be the sequence of the polypeptide MTBN1, MTBN2, MTBN3, MTBN4, MTBN5, MTBN6, MTBN7 or MTBN8 as depicted in FIGS. 1A and 1B, or a second amino acid sequence identical to the first amino acid sequence with conservative substitutions. The polypeptide has Mycobacterium tuberculosis specific antigenic and immunogenic properties. Also included in the invention is an isolated segment of this polypeptide, the segment being shorter than the full-length polypeptide and having Mycobacterium tuberculosis specific antigenic and immunogenic properties. Other embodiments are compositions containing the polypeptide, or functional segment, and a pharmaceutically acceptable diluent or filler. Compositions of the invention can also contain at least two (e. g., three, four, five, six, seven, eight, nine, ten, twelve, fifteen, or twenty) polypeptides of the Mycobacterium tuberculosis complex, or functional segments thereof, with at least one of the at least two (e. g., two, three, four, five, six, seven, or eight) polypeptides having the sequence of one of the above described polypeptides of the invention. The polypeptides will preferably be those not encoded by the genome of cells of the BCG strain of M. bovis.

The invention also features methods of diagnosis. One embodiment is a method involving: (a) administration of one of the above polypeptide compositions to a subject suspected of having or being susceptible to Mycobacterium tuberculosis infection; and (b) detecting an immune response in the subject to the composition, as an indication that the subject has or is susceptible to Mycobacterium tuberculosis infection. An example of such a method is a skin test in which the test substance (e. g., compositions containing one or more of MTBN1-MTBN8) is injected intradermally into the subject and in which a skin delayed-type hypersensitivity response is tested for. Another embodiment is a method that involves: (a) providing a population of cells containing CD4 T lymphocytes from a subject; (b) providing a population of cells containing antigen presenting cells (APC) expressing a major histocompatibility complex (MHC) class II molecule expressed by the subject; (c) contacting the CD4 lymphocytes of (a) with the APC of (b) in the presence of one or more of the polypeptides, functional segments, and or polypeptide compositions of the invention; and (d) determining the ability of the CD4 lymphocytes to respond to the polypeptide, as an indication that the subject has or is susceptible to Mycobacterium tuberculosis infection. Another diagnostic method of the invention involves: (a) contacting a polypeptide, a functional segment, or a polypeptide/functional segment composition of the invention with a bodily fluid of a subject; (b) detecting the presence of binding of antibody to the polypeptide, functional segment, or polypeptide/functional segment composition, as an indication that the subject has or is susceptible to Mycobacterium tuberculosis infection.

Also encompassed by the invention are methods of vaccination. These methods involve administration of any of the above polypeptides, functional segments, or DNA compositions to a subject. The compositions can be administered alone or with one or more of the other compositions.

As used herein, an “isolated DNA molecule” is a DNA which is one or both of: not immediately contiguous with one or both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally-occurring genome of the organism from which the DNA is derived; or which is substantially free of DNA sequence with which it occurs in the organism from which the DNA is derived. The term includes, for example, a recombinant DNA which incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Isolated DNA also includes a recombinant DNA which is part of a hybrid DNA encoding additional M. tuberculosis polypeptide sequences.

“DNA molecules” include cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the DNA molecule may be a sense strand or an antisense strand.

An “isolated polypeptide” of the invention is a polypeptide which either has no naturally-occurring counterpart, or has been separated or purified from components which naturally accompany it, e.g., in M. tuberculosis bacteria. Typically, the polypeptide is considered “isolated” when it is at least 70%, by dry weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.

Preferably, a preparation of a polypeptide of the invention is at least 80%, more preferably at least 90%, and most preferably at least 99%, by dry weight, the peptide of the invention. Since a polypeptide that is chemically synthesized is, by its nature, separated from the components that naturally accompany it, the synthetic polypeptide is “isolated.”

An isolated polypeptide of the invention can be obtained, for example, by extraction from a natural source (e.g., M. tuberculosis bacteria); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis. A polypeptide that is produced in a cellular system different from the source from which it naturally originates is “isolated,” because it will be separated from components which naturally accompany it. The extent of isolation or purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The polypeptides may contain a primary amino acid sequence that has been modified from those disclosed herein. Preferably these modifications consist of conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

The terms “protein” and “polypeptide” are used herein to describe any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). Thus, the term “Mycobacterium tuberculosis polypeptide” includes full-length, naturally occurring Mycobacterium tuberculosis protein, as well a recombinantly or synthetically produced polypeptide that corresponds to a full-length naturally occurring Mycobacterium tuberculosis protein or to particular domains or portions of a naturally occurring protein. The term also encompasses a mature Mycobacterium tuberculosis polypeptide which has an added amino-terminal methionine (useful for expression in prokaryotic cells) or any short amino acid sequences useful for protein purification by affinity chromatography, e.g., polyhistidine for purification by metal chelate chromatography.

As used herein, “immunogenic” means capable of activating a primary or memory immune response. Immune responses include responses of CD4+ and CD8+ T lymphocytes and B-lymphocytes. In the case of T lymphocytes, such responses can be proliferative, and/or cytokine (e. g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-13, IL-15, tumor necrosis factor-a (TNF-a), or interferon-y (IFN-y))-producing, or they can result in generation of cytotoxic T-lymphocytes (CTL). B-lymphocyte responses can be those resulting in antibody production by the responding B lymphocytes.

As used herein, “antigenic” means capable of being recognized by either antibody molecules or antigen-specific T cell receptors (TCR) on activated effector T cells (e. g., cytokine-producing T cells or CTL).

Thus, polypeptides that have “Mycobacterium tuberculosis specific antigenic properties” are polypeptides that: (a) can be recognized by and bind to antibodies elicited in response to Mycobacterium tuberculosis organisms or wild-type Mycobacterium tuberculosis molecules (e. g., polypeptides); or (b) contain subsequences which, subsequent to processing of the polypeptide by appropriate antigen presenting cells (APC) and bound to appropriate major histocompatibility complex (MHC) molecules, are recognized by and bind to TCR on effector T cells elicited in response to Mycobacterium tuberculosis organisms or wild-type Mycobacterium tuberculosis molecules (e. g., polypeptides).

As used herein, polypeptides that have “Mycobacterium tuberculosis specific immunogenic properties” are polypeptides that: (a) can elicit the production of antibodies that recognize and bind to Mycobacterium tuberculosis organisms or wild-type Mycobacterium tuberculosis molecules (e. g., polypeptides); or (b) contain subsequences which, subsequent to processing of the polypeptide by appropriate antigen presenting cells (APC) and bound to appropriate major histocompatibility complex (MHC) molecules on the surface of the APC, activate T cells with TCR that recognize and bind to peptide fragments derived by processing by APC of Mycobacterium tuberculosis organisms or wild-type Mycobacterium tuberculosis molecules (e. g., polypeptides) and bound to MHC molecules on the surface of the APC. The immune responses elicited in response to the immunogenic polypeptides are preferably protective. As used herein, “protective” means preventing establishment of an infection or onset of a disease or lessening the severity of a disease existing in a subject. “Preventing” can include delaying onset, as well as partially or completely blocking progress of the disease.

As used herein, a “functional segment of a Mycobacterium tuberculosis polypeptide” is a segment of the polypeptide that has Mycobacterium tuberculosis specific antigenic and immunogenic properties.

Where a polypeptide, functional segment of a polypeptide, or a mixture of polypeptides and/or functional segments have been administered (e.g., by intradermal injection) to a subject for the purpose of testing for a M. tuberculosis infection or susceptibility to such an infection, “detecting an immune response” means examining the subject for signs of an immunological reaction to the administered material, e.g., reddening or swelling of the skin at the site of an intradermal injection. Where the subject has antibodies to the administered material, the response will generally be rapid, e.g., 1 minute to 24 hours. On the other hand, a memory or activated T cell reaction of pre-immunized T lymphocytes in the subject is generally slower, appearing only after 24 hours and being maximal at 24-96 hours.

As used herein, a “subject” can be a human subject or a non-human mammal such as a non-human primate, a horse, a bovine animal, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a hamster, a rat, or a mouse.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Unless otherwise indicated, these materials and methods are illustrative only and are not intended to be limiting.

All publications, patent applications, patents and other references mentioned herein are illustrative only and not intended to be limiting.

Other features and advantages of the invention, e. g., methods of diagnosing M. tuberculosis infection, will be apparent from the following description, from the drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a depiction of the amino acid sequences of M. tuberculosis polypeptides MTBN1-MTBN8 (SEQ ID NOS:1-8, respectively).

FIGS. 2A-2E are a depiction of the nucleotide sequences of the coding regions (mtbn1-mtbn8) encoding MTBN1-MTBN8 (SEQ ID NOS:9-16, respectively).

FIG. 3 is a bar graph showing the delayed-type hypersensitivity responses induced by intradermal injection of 3 different test reagents in female guinea pigs that had been either infected with M. tuberculosis cells or sensitized with BCG or M. avium cells.

DETAILED DESCRIPTION

The genome of M. tuberculosis [Cole et al. (1998) Nature 393: 537-544] contains open reading frames (ORFs) that have been deleted from the avirulent BCG strain.

The polypeptides encoded by these ORFs are designated herein “M. tuberculosis BCG Negative” polypeptides (“MTBN”) and the ORFs are designated “mtbn.” The invention is based on the discovery that a MTBN polypeptide (MTBN4) elicited a skin response in animals infected with M. tuberculosis, but not in animals sensitized to either BCG or M. avium, a non-M. tuberculosis-complex strain of mycobacteria (see Example 1 below). These findings indicate that MTBN (e.g., MTBN1-MTBN8) can be used in diagnostic tests that discriminate infection of a subject by M. tuberculosis from exposure to both mycobacteria other than the M. tuberculosis-complex and BCG. The M. tuberculosis-complex includes M. tuberculosis, M. bovis, M. microti, and M. africanum. Thus they can be used to discriminate subjects exposed to M. tuberculosis, and thus potentially having or being in danger of having tuberculosis, from subjects that have been vaccinated with BCG, the most widely used tuberculosis vaccine. Diagnostic assays that are capable of such discrimination represent a major advance that will greatly reduce wasted effort and consequent costs resulting from further diagnostic tests and/or therapeutic procedures in subjects that have given positive results in less discriminatory diagnostic tests.

Furthermore, the results in Example 1 show that MTBN4, as expressed by whole viable M. tuberculosis organisms, is capable of inducing a strong immune response in subjects infected with the organisms and thus has the potential to be a vaccine.

The MTBN polypeptides of the invention include, for example, polypeptides encoded within the RD1, RD2, and RD3 regions of the M. tuberculosis genome [Mahairas et al. (1996) J. Bacteriol. 178: 1274-1282]. Of particular interest are polypeptides encoded by ORFs within the RD1 region of the M. tuberculosis genome. However, the invention is not restricted to the RD1, RD2, and RD3 region encoded polypeptides and includes any polypeptides encoded by ORFs contained in the genome of one or more members of the M. tuberculosis genome and not contained in the genome of BCG. The amino acid sequences of MTBN1-MTBN8 are shown in FIGS. 1A and 1B and the nucleotide sequences of mtbn1-mtbn8 are shown in FIGS. 2A-2E.

The invention encompasses: (a) isolated DNA molecules containing mtbn sequences (e.g., mtbn1-mtbn8) encoding MTBN polypeptides (e.g., MTBN1-MTBN8) and isolated portions of such DNA molecules that encode polypeptide segments having antigenic and immunogenic properties (i.e., functional segments); (b) the MTBN polypeptides themselves (e.g., MTBN1-MTBN8) and functional segments of them; (c) antibodies (including antigen binding fragments, e.g., F (ab′) 2, Fab, Fv, and single chain Fv fragments of such antibodies) that bind to the MTBN polypeptides (e.g., MTBN1-MTBN8) and functional segments; (d) nucleic acid molecules (e.g., vectors) containing and capable of expressing one or more of the mtbn (e.g., mtbn1-mtbn8) sequences and portions of DNA molecules; (e) cells (e.g., bacterial, yeast, insect, or mammalian cells) transformed by such vectors; (f) compositions containing vectors encoding one or more M. tuberculosis polypeptides (or functional segments) including both the MTBN (e.g., MTBN1-MTBN8) polypeptides (or functional segments thereof) and previously described M. tuberculosis polypeptides such as ESAT-6, 14 kDa antigen, MPT63, 19 kDa antigen, MPT64, MPT51, MTC28, 38 kDa antigen, 45/47 kDa antigen, MPB70, Ag85 complex, MPT53, and KatG (see also U.S. Pat. No. 6,087,163); (g) compositions containing one or more M. tuberculosis polypeptides (or functional segments), including both the polypeptides of the invention and previously described M. tuberculosis polypeptides such as those described above; (h) compositions containing one or more of the antibodies described in (c); (i) methods of diagnosis involving either (1) administration (e.g., intradermal injection) of any of the above polypeptide compositions to a subject suspected of having or being susceptible to M. tuberculosis infection, (2) in vitro testing of lymphocytes (B-lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes) from such a subject for responsiveness (e.g., by measuring cell proliferation, antibody production, cytokine production, or CTL activity) to any of the above polypeptide compositions, (3) testing of a bodily fluid (e.g., blood, saliva, plasma, serum, urine, or semen or a lavage such as a bronchoalveolar lavage, a vaginal lavage, or lower gastrointestinal lavage) for antibodies to the MTBN polypeptides (e. g., MTBN1-MTBN8) or functional segments thereof, or the above-described polypeptide compositions; (4) testing of a bodily fluid (e.g., as above) for the presence of M. tuberculosis, MTBN (e.g., MTBN1-MTBN8) polypeptides or functional segments thereof, or the above-described polypeptide compositions in assays using the antibodies described in (c); and (5) testing of a tissue (e.g., lung or bronchial tissue) or a body fluid (e.g., as above) for the presence of nucleic acid molecules (e.g., DNA or RNA) encoding MTBN polypeptides (e.g., MTBN1-MTBN8) (or portions of such a nucleic acid molecules) using nucleic acid probes or primers having nucleotide sequences of the nucleic molecules, portions of the nucleic molecules, or the complements of such molecules; and (j) methods of vaccination involving administration to a subject of the compositions of either (f), (g), (h) or a combination of any two or even all 3 compositions.

With respect to diagnosis, purified MTBN proteins, functional segments of such proteins, or mixtures of proteins and/or the functional fragments have the above-described advantages of discriminating infection by M. tuberculosis from either infection by other bacteria, and in particular, non-pathogenic mycobacteria, or from exposure (by, for example, vaccination) to BCG.

Furthermore, compositions containing the proteins, functional segments of the proteins, or mixtures of the proteins and/or the functional segments allows for improved quality control since “batch-to-batch” variability is greatly reduced in comparison to complex mixtures such as purified protein derivative (PPD) of tuberculin.

The use of the above-described polypeptide and nucleic acid reagents for vaccination also provides for highly specific and effective immunization. Since the virulent M. tuberculosis polypeptides encoded by genes absent from avirulent BCG are likely to be mediators of virulence, immunity directed to them can be especially potent in terms of protective capacity. Where vaccination is performed with nucleic acids both in vivo and ex vivo methods can be used. In vivo methods involve administration of the nucleic acids themselves to the subject and ex vivo methods involve obtaining cells (e.g., bone marrow cells or fibroblasts) from the subject, transducing the cells with the nucleic acids, preferably selecting or enriching for successfully transduced cells, and administering the transduced cells to the subject. Alternatively, the cells that are transduced and administered to the subject can be derived from another subject. Methods of vaccination and diagnosis are described in greater detail in U.S. Pat. No. 6,087,163, the disclosure of which is incorporated herein by reference in its entirety.

The following example is meant to illustrate, not limit the invention.

Example 1 MTBN4 Elicits a Specific Skin Reaction in Guinea Pigs Infected with M. tuberculosis

Four groups of outbred female guinea pigs (18 per group) were used to test the usefulness of the MTBN4 polypeptide as a M. tuberculosis-specific diagnostic reagent. The four groups were treated as follows.

Group 1 animals were infected by aerosol with approximately 100 M. tuberculosis strain H37Rv cells.

Group 2 animals were sensitized intradermally with 106 live M. bovis BCG Japanese cells.

Group 3 animals were sensitized intradermally with 106 live M. avium cells.

Group 4 animals were mock-sensitized by intradermal injection with saline.

Seven weeks after infection or sensitization, the animals were injected intradermally with 1 μg of PPD (6 animals from each group), 2 μg of purified recombinant MPT64 (6 animals from each group), or 2 μg of MTBN4 (6 animals from each group). The diameter of the resulting erythema was measured 24 hours later. Data are expressed as mean diameter of erythema (in mm) and standard deviations are indicated (FIG. 3).

No erythema was detected in the group 4 animals with any test substance and thus no data are shown for this group. On the other hand, group 1 animals (solid bars) showed a significant response with all three test substances. Group 2 animals (open bars) showed a significant response to PPD and MPT64 but not MTBN4.

Group 3 animals showed a significant response to PPD only (hatched bars).

Thus, PPD which contains antigenic/immunogenic molecules common to the M. tuberculosis-complex as well as other mycobacterial strains, gave the least discriminatory results in that it induced responses in animals infected with or sensitized to mycobacteria of the M. tuberculosis-complex (M. tuberculosis and BCG) as well as another non-pathogenic mycobacterium (M. avium).

While MPT64, which is encoded and expressed by both M. tuberculosis and BCG, did not elicit a response in animals infected with M. avium, it did elicit responses in both the M. tuberculosis infected and the BCG sensitized animals. Finally, MTBN4 elicited a response in only the M tuberculosis animals. Thus, it induced the most specific response and, most importantly, allowed for discrimination between animals infected with M. tuberculosis and those sensitized to BCG.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. 

What is claimed:
 1. A method of in vitro diagnosis that discriminates between infection by Mycobacterium tuberculosis-complex and vaccination by Bacille Calmette Guerin (BCG) strain of Mycobacterium bovis comprising: providing a population of cells comprising CD4 T lymphocytes from a subject; contacting cells of the population with at least two different antigens, wherein the antigens are isolated polypeptides of the Mycobacterium tuberculosis-complex that are not encoded by BCG, including at least one isolated polypeptide selected from the group consisting of (i) a first amino acid sequence comprising the sequence of MTBN4 (SEQ ID NO: 4), (ii) a second amino acid sequence that is an antigenic segment of MTBN4 and (iii) a third amino acid sequence that is identical to said first or second amino acid sequence but that has conservative substitutions and that retains Mycobacterium tuberculosis-complex specific antigenic properties; and determining whether or not there has been an immune response to said at least two different antigens, wherein CD4 T lymphocytes from a subject that has been infected by Mycobacterium tuberculosis-complex respond to said at least two different antigens, and CD4 T lymphocytes from a subject vaccinated with the BCG strain of Mycobacterium bovis but not infected by Mycobacterium tuberculosis-complex do not respond to said at least two different antigens.
 2. The method of claim 1, wherein said at least one isolated polypeptide comprises said second or third amino acid sequence.
 3. The method of claim 1, wherein the step of contacting is contacting said cells with a composition containing said at least two different antigens.
 4. The method of claim 3, wherein the determining step comprises testing for production of at least one cytokine.
 5. The method of claim 4, wherein the at least one cytokine includes interferon-γ (IFN-γ).
 6. The method of claim 1, wherein the determining step comprises testing for production of at least one cytokine.
 7. The method of claim 6, wherein the at least one cytokine includes interferon-γ (IFN-γ).
 8. The method of claim 1, wherein the isolated polypeptides of the Mycobacterium tuberculosis-complex are encoded within the RD1, RD2, and RD3 regions. 